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Yeast system capable of coexpressing CYP and CPR

A co-expression and yeast technology, applied in the field of drug-metabolizing enzyme heterologous expression system, can solve problems such as unstable protein expression, low data repeatability, and system instability, and achieve the effects of easy construction, stable system, and reduced production costs

Inactive Publication Date: 2010-02-24
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are still the following disadvantages when Saccharomyces cerevisiae system is used for co-expression of CYP enzymes: 1. Co-expression with hydrogen donor CPR can increase the activity of CYP, but it is easy to cause system instability; 2. The vectors expressing CYP and CPR are too Large, the loss of the carrier in the system is serious, and the protein expression is unstable, which leads to low data reproducibility in the in vitro drug metabolism evaluation of the system

Method used

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  • Yeast system capable of coexpressing CYP and CPR
  • Yeast system capable of coexpressing CYP and CPR
  • Yeast system capable of coexpressing CYP and CPR

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1 Construction and Identification of Yeast Expression Plasmids

[0044] 1. Extract yeast chromosome and amplify to obtain PGK fragment. The obtained PGK amplified fragment was recovered and purified and directly ligated into the cloning vector pGEM-T easy vector, and the sequence of the obtained recombinant was confirmed to be correct. The vector pT-PGK uses Klenow enzyme to remove the NdeI site to obtain the pT-PGKn plasmid. LEU screening gene and pT-PGKn were digested by SacII and SphI, and then by T 4 DNA ligase connection, transformed into Escherichia coli, the resulting recombinant is the pTPL plasmid (such as figure 1 ). Using pTPL as a template, primers were designed for PCR amplification. Obtained pTPL' fragment, the length is about 5000bp (comprising the sequence of yeast promoter, terminator, yeast selection gene, Escherichia coli cloning vector).

[0045] 2. hCPR cDNA and hCYP1A2, hCYP2D6, hCYP2E1, hCYP2C9, hCYP3A4 cDNA were obtained by PCR ampl...

Embodiment 2

[0046] Example 2: Construction and identification of yeast expression system

[0047] The plasmid pTPL-hCPR was linearized with SphI restriction endonuclease, and the yeast BWG1-7α competent cells were transformed. The recombinant was identified by leucine screening, and the host strain BWG-CPR was constructed. Yeast chromosomes were extracted and PCR amplified to identify recombinants. At the same time, yeast microsomes were prepared, microsomes were electrophoresed by SDS-PAGE, and CPR expression bands were analyzed by western blot (such as figure 2 ).

[0048] The plasmid pYeplac195-CYP was directly transformed into BWG-CPR competent cells, and the recombinant was screened and identified with uracil, and the strain BWG-CPR / CYP was constructed. Pick transformed colonies and inoculate them in liquid YPD medium, and culture them at 30°C until OD 600 =0.6-0.8, yeast chromosomes were extracted and PCR amplified to identify recombinants. At the same time, yeast microsomes we...

Embodiment 3

[0049] Example 3: Expression of protein in yeast and analysis of system activity and stability

[0050] 1. Preparation of yeast microsomes (yeast microsome, YM)

[0051] Fermentation medium 1L, medium components: 10g of yeast extract, 20g of peptone, 30g of glucose, distilled in 1000ml of distilled water.

[0052] When the cells start to shake, add cell δ-ALA to make the final concentration 0.5mmol / L. Shake at 30°C for 6 hours, cool down to 25°C, shake for 30 minutes, then rapidly raise the temperature to 30°C until the cells grow to OD 600 =1.0-1.5, centrifuge the bacterial liquid, collect the bacterial cells, and prepare yeast microsomes.

[0053] 2. Activity determination and stability analysis of yeast system

[0054] Use the Bradford method to detect the protein content of yeast microsomal, and detect the CPR activity (Table 4) and CYP content (such as Figure 4 , CYP3A4-CO reduction absorption peak). The expression levels of hCYP1A2, hCYP2D6, hCYP2E1, hCYP2C9, and h...

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Abstract

This invention relates to a drug-metabolizing enzyme heterogenesis expression system used for out-of-body drug metabolism property evaluation, exactly to say a yeast expression system of coexpressingCYP and CPR, the yeast expression vector which respectively has CPR DNA fragment and CYP DNA fragment is transduced into yeast cell, this two expression vector express in yeast cell by means of occlusal pattern and liberation respectively , custom-crafted yeast cell is fermented and induced, getting yeast cell product which is purpose expression system, it can be used for CYP enzyme system out-of-body drug metabolism property evaluation. The constructed new Saccharomyces cerevisia co-expression system can be extensively used for oxidation reduction enzyme system such as cytopigment P450 enzymesystem, the latter one is extensively used for screening, application and development of drug metabolism involved in ADME / T and pharmacokinetics.

Description

technical field [0001] The present invention relates to a drug metabolism enzyme heterologous expression system for in vitro drug metabolism property evaluation, specifically a stable co-expression of human cytochrome P450 enzyme (CYP) and human cytochrome P450 oxidoreductase (CPR) A new expression system for Saccharomyces cerevisiae. Background technique [0002] At present, the preclinical ADME / T and pharmacokinetic research has changed from the traditional model of single experimental animal as the research object to the comprehensive molecular level, protein level, cell level, tissue and organ level, etc. An evaluation model or system for the similarities and differences of ADME / T properties of animals. Among them, the evaluation of drug metabolism properties using in vitro recombinant human metabolic system is conducive to reducing inter-species errors, improving accuracy and greatly shortening research and development time. Using molecular biology methods, using huma...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/19C12N15/81C12N15/52C12N15/53C12R1/865
Inventor 杨凌程婕
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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